Mouse Models of Inflammatory Lung Diseases
Zhou Zhu, MD, Ph.D.
Division of Allergy and Clinical Immunology
Johns Hopkins University School of Medicine
Outlines
• Basic lung structure, function, and inflammatory lung diseases
• Methods, techniques, and approaches to studying lung diseases
• Mouse models of inflammatory lung diseases
Lung: Basic Structure
Anatomy
From Wikipedia
1
Lung: Basic Structure
Lung Histology
H&E (100x)
Airways
Alveoli
Blood
Vessel
Lung Function and Diseases
• Gas exchange: O2 from atmosphere to bloodstream and CO2
from bloodstream to atmosphere
• Diseases
• COPD
• Asthma
• Pneumonia
• Pulmonary fibrosis
• Sarcoidosis
• Cystic fibrosis
• Lung cancer
• …
Studying the Lung: Methods and Techniques
• Pulmonary Function Test (PFT)
• Airway resistance
• Lung compliance
• Bronchoalveolar Lavage (BAL)
• Inflammatory cells in the airway: total and differential
• Mediators: cytokines, chemokines
• Lung tissues
• Histology: H&E, Alcian Blue, PAS, Trichrome
• Proteins
• RNA
• Immunohistochemistry (IHC)
• FACS analysis of cell populations
• Culture of specific cell types
2
Studying the Lung: Approaches
• Stimulation
• Allergens
• Cigarette smoke
• Transgenics
• Inflammatory cytokines
• Knock-out & knock-in
• Cytokines & receptors
• Siglecs
• Mutant strains
• Phosphatases
Allergen Induced Allergic Asthma
Protocol for ovalbumin (OVA) induced allergic asthma
Day 0
Day 7
Immunization with OVA
Day 14
Day 15
Day 16
Intranasal
50 µg OVA
Day 18
PFT & Sacrifice
i.p. injection
50 µ g OVA / 1 mg alum in 200 µ L
OVA Induced Airway Inflammation
BAL Cells
Macrophages
Eosinophils
PBS
OVA
3
OVA Induced Airway Inflammation
Differential Cell Counts
cell differentiation(%)
40
30
20
10
120
PBS/WT
100
OVA/WT
80
60
40
20
0
Ly
m
Eo
s
N
eu
/W
VA
O
S/W
PB
M
ac
T
0
T
BAL Total cell number (X10 4 )
Total Cell Counts
OVA Induced Lung Inflammation
H&E (100x)
PBS
OVA
OVA Induced Lung Inflammation
H&E (400x)
PBS
OVA
4
OVA Induced Lung Inflammation
Alcian Blue (100x)
PBS
OVA
OVA Induced Lung Inflammation
Alcian Blue (400x)
PBS
OVA
OVA Induced Lung Inflammation
Trichrome (100x)
PBS
OVA
5
OVA Induced Lung Inflammation
Trichrome (400x)
PBS
OVA
Elastase Induced Emphysema
Protocol for elastase induced emphysema
Porcine Pancreatic Elastase
1u/mouse, I.T. once
0 1 2 3
Day
7
14
21
28
Sacrifice &
Collect BAL, lung tissue
Elastase Induced Airway Inflammation
BAL Cells
Day 2
Day 21
PBS
6
Elastase Induced Emphysema
Lung Histology (H&E 20x)
Day 3
Day 21
SHP-1
Src Homology 2 domain-containing protein tyrosine
Phosphatase-1
Encoded by the hcp gene
Primarily expressed in hematopoietic-derived cells
Negative regulator of a variety of growth factor/cytokine
signaling pathways
Activated SHP-1 → binds to its target proteins → dephosphorylates the phospho-tyrosine residues → terminates the
signaling
SHP-1 Regulation of Signaling
N
SH2 ITIM
SH2
SH2
SH2
N
ITIM
C
P Subs
Subs
P
C
Inactive
Active
7
SHP-1 Deficiency in Mice
Motheaten mice (me/me) – a natural mutant strain
Viable motheaten mice (mev/mev) – another spontaneous mutant strain
⇒ The mutations cause aberrant splicing of the SHP-1 transcript,
resulting in diminished production of functional SHP-1 protein
Phenotype
inflammatory lesions of the skin, increased neutrophils in the
peripheral blood, glomerularnephritis, abnormal lymphoid
tissues, pneumonitis with macrophage infiltration in the
alveoli, increased production of immunoglobulins, depressed
immune responses, profound NK-cell deficiency, and a
shortened life span (average 22 days and 61 days, respectively)
IL-4/IL-13 Signaling Systems
IL-4/IL-13
Jak-1
IL-13Rα1
α1
Cell Growth
& Survival
IRS-1/2
IL-4Rα
α
IL-4Rα
α
γc
Tyk-2
PI3K
STAT-6
IL-4
IL-4/IL-13
Responsive Genes
SHP-1
SHIP-1
SOCS
Type I
Type II
SHP-1 Expression and Activation in Lung Epithelial Cells
MLE12
TNF-α
α
Time (min): 0 3
5
10 30
IL-13
5 10 30
p- SHP-1
SHP-1
Mouse Lung
α-SHP-1
IHC: Control IgG
A
α-SHP-1
B
C
8
Hypothesis
SHP-1 is an important regulator of
• Immunological homeostasis of the lung
• Allergic inflammatory responses
SHP-1 Deficiency and Phenotype
mev/+: Phenotypically Normal
mev/mev: Inflammatory phenotype
Pulmonary Inflammation
WT
mev
100
*
WT
mev
80
10x
*
60
10x
mev
mev
40
*
20
*
*
Lym
Neu
0
Total
40x
Mac
Eos
100x
9
Mucus Hyperproduction
A.
B.
PAS
Alcian Blue
RT-PCR
WT
WT
mev
WT
mev
MUC5AC
β -Actin
Immunoblot
mev
MUC5AC
Pulmonary Fibrosis
WT
RT-PCR
ELISA
BAL TGF-β
β 1 (pg/ml)
Trichrome
mev
WT
mev
TGF-β
β1
β -Actin
*
450
WT
mev
400
350
300
250
*
200
150
100
50
0
Total Activated
Airway Resistance and Hyperresponsiveness
14
WT (n=4)
12
mev (n=9)
RL (cmH2O/ml/s)
10
8
6
*
*
*
*
4
2
0
0
2
4
6
Methacholine (mg/ml)
8
10
12
14
(FlexiVent System)
10
Upregulation of Th2 Cytokines
RT-PCR
ELISA
mev
WT
IL-4
70
WT
60
meV
*
50
IL-5
40
30
IL-13
*
*
20
IFN-γγ
10
β -Actin
0
IL-4
IL-5
IFN-γγ
IL-13
Role of Th2 Cytokines in Pulmonary Inflammation
mev/IL-13KO
H&E
mev/STAT6KO
H&E
BAL Cellularity
BAL Cellularity
180
90
70
80
60
*
40
Differential x104
IL-13KO
Total Cells x104
WT
60
Macrophage
Eosinophil
Neutrophil
Lymphocyte
300
250
50
WT
*
40
30
SHP-1
IL-13
+/+
+/+
+/+
-/-
-/+/+
mev/IL-4KO
-/-/-
* *
*
0
SHP-1
IL-13
200
150
Macrophage
Eosinophil
Ne utrophil
Lymphocyte
120
90
150
*
60
100
* *
*
30
10
0
mev/IL-13KO
STAT6KO
20
20
mev
Differential x10 4
80
100
Total Cells x10 4
120
+/+
+/+
+/+
-/-
-/+/+
50
0
0
-/-/-
mev
mev/STAT6KO
mev/IL-13KO
SHP-1 +/+
STAT-6 +/+
+/+
-/-
-/+/+
-/-/-
SHP-1
STAT-6
+/+
+/+
+/+
-/-
-/+/+
-/-/-
mev/STAT6KO
Increased Mast Cells in the Lung
WT
x100
mev
x100
11
Histamine Content and Release
Histamine (ng/mL)
*
Spleen
WT
(1x10 6 cells)
mev
0.4
0.3
0.5
Lung
(1x10 6 cells)
Histamine (ng/mL)
0.5
*
0.2
*
0.1
0.0
*
0.4
0.3
*
0.2
0.1
0.0
Total
Spontaneous PMA/Ionomycin
Total
Spontaneous
Cytokine Production by Splenocytes
IL-13
MIP-1 α
IL-4
100
35
400
90
30
350
(pg/ mL)
80
300
25
70
20
60
250
50
200
15
40
Anti-FcεRIα
100
20
5
50
10
0
0
WT
mev
0
WT
mev
10
120
mev
IL-12
140
9
100
WT
IFN-γγ
IP-10
(pg/ mL)
Medium
150
30
10
120
8
100
7
80
6
Medium
80
Anti- FcεRIα
5
60
40
4
60
3
40
2
20
20
1
0
0
WT
mev
0
WT
mev
WT
mev
Hexosaminidase Content and Release by BMMC
% Hexosaminidase Released from BMMC
Hexosaminidase (%)
400
350
WT
300
mev
250
200
150
100
50
0
Total
Blank
PMA/ Ionomycin
12
Mast Cells and Lung Inflammation
Crossbreeding to mast cell deficient Kw-sh mice
C57BL/6
KW-sh
mev
mev x KW-sh
Summary
mev mice:
• Spontaneous and progressive pulmonary inflammation
rich in macrophages, lymphocytes, and eosinophils
↑ Th2 cytokines IL-4, IL-5, and IL-13
↑ Th2 chemokines Eotaxin and MCP-1
• Mucus hyperplasia ↑ Mucin genes
• Subepithelial and parenchymal fibrosis ↑ TGF-β
• Increased airway resistance and AHR
• Mast cell-dependent
13